Density Of Varicosities Research Articles

Compartmental ordering of cholinergic innervation in the mediodorsal nucleus of the thalamus in human brain

The cholinergic innervation of the mediodorsal (MD) nucleus of the thalamus was visualized immunohistochemically in human brain postmortem, using an antibody against human choline acetyltransferase (ChAT). The ChAT staining of the MD nucleus was more intense than in the surrounding thalamic nuclei but weaker than that of the striatum. No ChAT-positive cell bodies were detected. The ChAT-positive neuropil was unevenly distributed, with patches of dense immunoreactivity with a weaker surrounding matrix. In adjoining sections stained for ChAT immunoreactivity and for acetylcholinesterase (AChE) activity, the zones enriched in AChAT-immunostained neuropil corresponded to AChE-rich regions. The three-dimensional reconstruction of the richest zone in AChE/ChAT activity evidenced a cylindrical organization throughout the rostrocaudal axis of the MD nucleus. Counts of ChAT-positive varicosities confirmed an inhomogeneous Distribution; the density of varicosities was 30% higher in ChAT-rich regions than in surrounding matrix. These findings suggest that the activity of intrinsic neurons within the nucleus may be differentially regulated by cholinergic systems.

Origin of noradrenergic innervation of the spleen in rats

Noradrenergic (NA) sympathetic innervation of the spleen was examined in young adult Sprague-Dawley rats (200–250 g) following surgical removal of the superior mesenteric-celiac ganglia (SM-CG) and/or bilateral transection of the subdiaphragmatic vagus nerve. Sham-operated and unoperated rats served as controls. NA sympathetic innervation of spleens from sham-operated and unoperated controls, and from vagotomized rats, was qualitatively similar, with fibers distributing to the capsule, trabeculae, vasculature, and parenchyma of the white pulp. Complete ganglionic extirpation resulted in almost total denervation of NA fibers in all compartments of the spleen. High-performance liquid chromatography with electrochemical detection (LCEC) for catecholamines (CA) and quantitative morphometry of the density of NA varicosities confirmed these observations. LCEC revealed a greater than 85% depletion of norepinephrine (NE) in the spleen following superior mesenteric-celiac ganglionectomy. Stereological evaluation of NA varicosities with a point counting method revealed a decline of 99% in the volume density of NA terminals that occurred uniformly in all compartments of spleens from ganglionectomized rats. In addition, stereological analysis revealed a loss of total NA varicosities (approximately 31% decrease) in spleens from sham-operated rats. This loss in volume density occurred largely due to a loss in parenchymal fibers (approximately 45% decrease). Bilateral subdiaphragmatic vagotomy blocked the effect on NA innervation produced by the surgical stress of sham operation. Retrograde tracing following injection of either fluorogold or true blue into the spleen, coupled with immunocytochemical localization of tyrosine hydroxylase (TH), demonstrated abundant fluorogold (true blue)-labeled neurons in the SM-CG; many, but not all, of these neurons also were TH-positive. These findings indicate that the SM-CG neurons supply NA innervation to the spleen, providing sympathetic innervation as the second neuron in the classical two-neuron sympathetic chain, and suggest additional non-NA innervation of the spleen as well. This study also suggests that surgical stress of sham operation may alter directly the NA innervation of the spleen, possibly by inducing temporary retraction of NA fibers of the parenchymal compartment, which is likely to reduce the availability of NE for interaction with cells of the immune system that possess adrenoceptors and are present adjacent to NA varicosities in this region. Bilateral vagotomy ameliorated the effects of sham operation on NA innervation; since the vagal nerve does not distribute fibers to the spleen, this effect is likely to occur through altered feedback circuits effecting sympathetic outflow, or through altered neuroendocrine outflow.

Simultaneous catecholamine histofluorescence and thymidine autoradiography of the sexually dimorphic nucleus of the preoptic area in the rat

The sexually dimorphic nucleus of the preoptic area (SDN-POA) in the rat represents a morphological substrate in which the influence of gonadal hormones on the process of sexual differentiation of the brain can be seen. Since the medial preoptic area (MPO) is a region rich in catecholamine (CA) terminals, it is possible that catecholamines may play a role either in the differentiation of the perinatal SDN-POA or in the function of this nucleus in the adult. It is not known whether catecholamine terminals exist within the SDN-POA or whether they can directly influence the activity of SDN-POA neurons. The present study was conducted to determine the extent to which catecholamines innervate this nucleus and further to elucidate the possibility of a potential sexual dimorphism in the innervation pattern. In order to determine which of the neurons in the MPO are within the SDN-POA we have utilized the fact that the SDN-POA has a prolonged period of neurogenesis in comparison to other neurons of the MPO. Thus, tritiated thymidine-labeled neurons can be used as a detection criterion for the SDN-POA. To conduct this experiment, timed pregnant Sprague-Dawley females were given a single injection of [ 3H]thymidine on Day 18 of gestation. Pups were killed as adults and prepared for fluorescence histochemistry of monoamines. Sections adjacent to those examined for catecholamine fluorescence were treated for autoradiographic localization of [ 3H]thymidine. Fluorescence innervation patterns were plotted within the boundaries of the nucleus following its identification from Nissl sections as well as from adjacent autoradiograms simultaneously viewed in a comparator bridge microscope with dark-field illumination. A low density of CA varicosities was seen within the SDN-POA of male rats; the density of varicosities was significantly greater, as measured stereologically, in the female. In each sex, varicosities were seen in apposition to the neurons of the SDN-POA. In consideration of the generally low density of varicosities in the associated neuropil, it is noteworthy that CA varicosities appeared in apposition to neuronal perikarya of the SDN-POA. Thus, the SDN-POA appears to receive a sexually dimorphic catecholamine innervation which can be revealed by the simultaneous analysis of birthdating and histofluorescence using a single histochemical preparation.

A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord

While several peptides have been shown to coexist in perikarya within dorsal root ganglia of rat, coexistence of peptides has not been confirmed in axons associated with these neurons. In this study, the coexistence of substance P (SP) with somatostatin (SOM), calcitonin gene-related peptide (CGRP), dynorphin A 1-8 (DYN), neurotensin (NT), galanin (GAL), and 5-HT in varicosities was visualized using fluorescence immunohistochemistry. Densities of immunoreactive varicosities within laminae I and II of the dorsal horn of the rat spinal cord were quantified by computer-assisted image analysis. Decreases in densities of immunoreactive varicosities as a result of multiple unilateral dorsal rhizotomies were used to determine proportions of immunoreactive varicosities associated with primary afferent neurons. Three observations were made. (1) Dorsal rhizotomy depleted greater than one-third of the varicosities individually immunoreactive for SP, SOM, GAL, or DYN, confirming the association of these peptides with primary afferent neurons. (2) SP coexisted with CGRP, GAL, and DYN in varicosities within the dorsal horn of normal animals. (3) CGRP-, SP+CGRP-, and SP+GAL-immunoreactive varicosities were nearly depleted following dorsal rhizotomy. The depletion of these peptides, particularly in combination, indicates that they may be used as markers for varicosities of some primary afferent neurons within the superficial laminae of the dorsal horn of the rat spinal cord.

Quantitative morphometric analysis of two types of serotonin-immunoreactive nerve fibres differentially responding to p-chlorophenylalanine treatment in the rat brain

Two types of serotonin neurons differentially responding to treatment with p-chlorophenyl-alanine, a potent inhibitor of serotonin synthesis, have been demonstrated in the rat brain by immunohistochemistry using a serotonin monoclonal antibody in combination with a quantitative morphometric analysis. In control rats, the numerical density of serotonin-immunoreactive varicosities and terminals varied considerably from region to region. In p-chlorophenylalanine-treated rats, the numerical density was reduced in every region, but the degree of reduction in each region differed markedly. No correlation was seen in values of the numerical density between the control and p-chlorophenylalanine-treated rats, indicating that serotonin-positive axons in p-chlorophenylalaninetreated rats were present independent of the degrees of the axon density seen in the controls. It is postulated, therefore, that there are at least two types of positive axons; one highly sensitive to p-chlorophenylalanine treatment and another relatively resistant to it. Since the numerical density in any region was always smaller in p-chlorophenylalanine-treated rats than in the controls, p-chlorophenylalanine-sensitive axons appeared to be more or less distributed in every region where positive axons were detectable under normal conditions. However, a number of regions were innervated purely by p-chlorophenylalanine-sensitive axons. By contrast, p-chlorophenylalanine-insensitive axons were observed in several definite regions. These regions appeared to be related to the limbic system, or cranial motor and parasympathetic nuclei. In the latter nuclei, p-chlorophenylalanine-insensitive axons seemed to possess such common morphological features as having relatively large varicosities and being situated adjacent to neural somata, suggesting their inhibitory roles in neurotransmission. The present result indicates that the two types of serotonin axons may play important roles in the behavioural events observed in rats following p-chlorophenylalanine treatment.

Somatostatin immunoreactive structures in the developing rat spinal cord

Somatostatin immunoreactive (SOM-I) perikarya were first observed in the ventral horn at E12, in the presumptive intermediate gray area at E14, and in the alar plate of the rostral spinal cord at E14. In general, after their initial appearance, their density increased and then decreased during development. A moderate density of SOM-I varicosities became obvious in the superficial laminae of the E20 dorsal horn. By E12 a few SOM-I perikarya, interpreted to be dorsal root ganglia, were observed lateral to the spinal cord, and by E13, SOM immunoreactivity was visualized within the central and peripheral processes of dorsal root ganglion axons. In the marginal zone, SOM-I fibers were first demonstrable in the ventral funiculus at E14, and in the lateral funiculus at E15. After their initial appearance, their density increased and then decreased with age, with the exception of the dorsal part of the lateral funiculus where it increased at the early stages of development to an apparently stable level. The early detection of SOM immunoreactivity in specific spinal regions corresponds well with the birth dates of cells in those regions. This indicates that the SOM-I cells are capable of synthesizing the substance at least as early as they have entered their final cell division.

Developmental changes in density and distribution of serotoninergic fibers in the chick spinal cord.

Developmental changes of serotoninergic innervation in the chick spinal cord (third lumbosacral segment) were examined with an immunohistochemical technique using an antiserum to serotonin. In the 1-day-old hatched chick, serotoninergic fibers were located in laminae I, II, VII, IX, and X. A large number of serotonin-positive fibers and terminals were found around somal profiles of large neurons and in the neuropil of the medial and lateral parts of the lateral motor column (LMC). In the 1-week-old chick, the density of serotoninergic fibers was greatly increased in the posterior columns, and serotoninergic fibers were most densely aggregated in the dorsolateral part of the LMC. In the 2-week-old chick, a considerable decrease in the density of serotoninergic fibers was observed in the lateral funiculus and the gray matter (laminae I, II, VII, IX, and X). In the LMC, serotonin-positive fibers and terminals were largely absent from the neuropil, but were found preferentially around the somal profiles of large neurons. Between 1 and 2 weeks after hatching the density of varicosities and terminals in the neuropil of the dorsolateral and medial parts of the LMC decreased by 33% and 56%, respectively. In the 3-month-old chick, the density of serotoninergic fibers in laminae I, II, V, VII, and X had increased compared to younger ages. Serotonin-positive fibers were not evenly distributed in the LMC of the adult chicken; rather, they were densely aggregated around the soma and proximal dendrites of motoneurons in the dorsolateral LMC. Many neuronal soma in the medial and intermediate regions of the LMC lacked serotoninergic fibers.

Substance P and cholecystokinin-like immunoreactive varicosities in somatosensory and autonomic regions of the rat spinal cord: a quantitative study of coexistence

Substance P- and cholecystokinin-like immunoreactivities have been shown to coexist in some, but not all, dorsal root ganglion cell bodies of the rat. Quantitative immunofluorescence techniques were used in the present study to describe densities of substance P- and cholecystokinin-like immunoreactive varicosities in several spinal cord nuclei. By combining simultaneous immunofluorescent techniques on one tissue section with computerized image processing, coexistence of substance P- and cholecystokinin-like immunoreactivity in varicosities was also quantified. By comparing spinal cord regions between normal and unilateral dorsal rhizotomy affected animals, densities of substance P- and cholecystokinin-like immunoreactive varicosities associated with primary afferent neurons were established. To determine the densities of immunoreactive varicosities that were related to unmyelinated primary afferent fibers, data were compared between normal animals and those treated neonatally with capsaicin. Four major observations were made: (1) Substance P- and cholecystokinin-like immunoreactivity coexist in populations of varicosities in sensory and autonomic regions of rat spinal segment L6. (2) Within the superficial laminae of the dorsal horn, varicosities containing both substance P- and cholecystokinin-like immunoreactivity and cholecystokinin-like immunoreactivity alone are of primary afferent neuron origin, but those containing only substance P-like immunoreactivity are most likely of spinal or descending neuronal origin. (3) Capsaicin-insensitive cholecystokinin-like immunoreactive varicosities were present predominantly in lamina I. These data suggest some cholecystokinin-like immunoreactive varicosities are associated with myelinated primary afferent neurons. (4) Primary afferent fibers containing substance P- and cholecystokinin-like immunoreactivity project to intermediate gray regions of the rat spinal cord. A large proportion of these fibers are capsaicin sensitive, suggesting that they are unmyelinated.

Localization of FMRFamide-like peptides in the snail Helix aspersa.

The distribution of FMRFamide-like material in the gastropod mollusc, Helix aspersa, was studied by radioimmunoassay (RIA) and immunocytochemistry. Most of the RIA activity was concentrated in the central nervous system, the male reproductive tract, the tentacles and the posterior digestive system (Table 1). The density of FMRFamidergic perikarya, nerves and nerve varicosities in the muscle tissue of all these regions, as indicated immunocytochemically (Fig. 2), was well correlated with the distribution as determined by RIA. Gel chromatography of each extract resolved two peaks of FMRFamide-like immunoreactivity (Fig. 3). The first of these was further analysed by high-pressure liquid chromatography (HPLC), and the components included two major immunoreactive peaks identifiable, both by their retention times and their effects on the radula protractor muscle of Busycon contrarium, as the known peptides FMRFamide and pQDPFLRFamide (Figs 4-6). The second peak from gel chromatography gave only a single peak, distinct from that of FMRFamide and pQDPFLRFamide, in two HPLC systems (Fig. 7), but it did not behave like a competitive ligand in the FMRFamide RIA. Moreover, its immunoreactivity, unlike any peptides we tested, was not affected by carboxypeptidase Y (Fig. 8), and it was not active on the radula protractor muscle. Thus, it is certainly not an FMRFamide-like peptide. We conclude that Helix aspersa contains at least two FMRFamide-like peptides, FMRFamide and pQDPFLRFamide. These peptides appear to act both as neurohormones and as neurotransmitters or modulators in the central ganglia, reproductive, digestive, muscular and circulatory systems.

Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: II. Pre- and postnatal development.

The pre- and postnatal development of cholinergic projections was investigated in the cat striate cortex by applying immunohistochemical methods based on a monoclonal antibody against choline acetyltransferase (ChAT). The earliest age investigated was gestational day 54. At this stage a sparse network of ChAT(+) fibers was distributed throughout the striate cortex. Subsequent postnatal maturation of ChAT(+) fibers was characterized by an increase in fiber density that started in layer VI and gradually progressed toward more superficial layers. By 4 weeks of age the density of ChAT(+) fibers and varicosities had reached adult levels in layers V and VI but was still subnormal in layers I-IV. The mature pattern of cholinergic innervation was established by 13 weeks of age. There was no evidence for developmental gradients in the anteroposterior and mediolateral directions within area 17. These results indicate that the cholinergic projection to striate cortex develops continuously in an inside-out sequence as is characteristic for most cortical maturation processes. There was no indication that striate cortex receives an especially dense cholinergic input during the critical period.

Choline acetyltransferase immunoreactivity in the rat thalamus.

The distribution of choline acetyltransferase immunoreactivity in the rat thalamus was investigated by using a specific monoclonal antibody and was compared with the pattern of acetylcholinesterase staining. The only choline acetyltransferase-immunoreactive cell bodies in the thalamus were in the medial habenula. A wide range of densities of immunoreactive fibers and varicosities was found. The highest densities of stained varicosities were in the anteroventral, reticular, lateral mediodorsal, and intralaminar nuclei. At the other extreme, the anterodorsal, ventroposteromedial, and paraventricular nuclei were almost devoid of immunoreactive varicosities. A light density of fibers was observed in several medial nuclei, including parataenial, reuniens, and gelatinosus. Most other nuclei contained moderately dense regions of varicose fibers that were often heterogeneous or patchy. The pattern of choline acetyltransferase immunoreactivity in the thalamus was in general similar to that of acetylcholinesterase. A marked discrepancy, however, was found in the anterodorsal nucleus, which was intensely stained for acetylcholinesterase but contained no apparent choline acetyltransferase immunoreactivity. Numerous physiologic studies have demonstrated striking effects of acetylcholine on thalamic activity. The present study provides a description of choline acetyltransferase-immunoreactive structures in the thalamic nuclei, providing a first step toward elucidating the anatomical basis for the physiologic and functional importance of cholinergic transmission in the thalamus.

Alterations of monoamines in specific central autonomic nuclei following immunization in mice

At the peak of an immune response (Day 4 following immunization) in mice, norepinephrine (NE) was selectively decreased in the paraventricular nucleus (PVN) of the hypothalamus. At times before (Day 2) and after (Day 8) the peak immune response, no changes in NE were found in this nucleus. Decreases in NE were not seen in other hypothalamic sites or in the Al cell group of the medulla, which sends noradrenergic projections to the hypothalamus, suggesting that the effect is selective and regional. Morphometric evaluation of varicosities revealed no alterations in density of catecholamine-containing varicosities in the PVN, further suggesting that the decrease in NE is a metabolic effect and not a loss or redistribution of fibers. NE also was decreased in the hippocampus on Day 2 at the rising phase of the immune response. In addition, alterations in serotonin levels were found in the brain during an immune response. Serotonin was decreased in the hippocampus on Day 2, was decreased in the PVN and supraoptic nucleus on Day 4, and was increased in the nucleus tractus solitarius (NTS) on Day 2. These results support the presence of a functional link from the activated immune system to central autonomic nuclei interconnecting the hypothalamus, the limbic system, and the autonomic nervous system. In view of the role of the PVN in corticotropin-releasing factor secretion and regulation of autonomic outflow, evidence from lesion studies for hippocampal involvement in immune regulation, and a key role for NTS in regulation of autonomic outflow, the present findings reinforce the potential importance of these brain regions in reciprocal communication between the nervous and immune systems.

Powering of bulk transport (varicosities) and differential sensitivities of directional transport in growing axons

Goldfish retinal ganglion cell (RGC) axons, regenerating in vitro, have varicosities, intervening phase-dense inclusions (IPDIs) and particles that are mobile. Varicosities contain an aggregate complex of cytomembranes embedded in a cytoskeletal matrix, and, when they saltate, they represent a form of bulk transport. While movement of varicosities is normally infrequent, the incidence of movement can be greatly increased by alkalinization with NH 4Cl. However, alkalinization also lowers the phase density of varicosities to reveal that motile hyperdense particles appear to be responsible for powering the translocation of varicosities and IPDIs. Other effects of alkalinization include a selective arrest of all anterograde movements and approximately a 10-fold reduction in the rate of retrograde mobility of particles and IPDIs. In mildly permeabilized axons, 20 μM orthovanadate selectively arrests retrogradely directed particle movements, while 100 μM arrests both antero- and retrograde transport. In addition to demonstrating in RGC axons that antero- and retrograde mechanisms exhibit differential pharmacological and pH sensitivities, the observations indicate that a heterogenous bulk mass can be translocated in growing axons by a passive ‘piggyback’ mechanism.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the guinea pig heart.

The distribution of calcitonin gene-related peptide like-immunoreactivity (CGRP-IR) within the heart and adjacent blood vessels of the guinea pig was investigated immunohistochemically by use of the peroxidase-antiperoxidase (PAP) technique. Numerous paravascular and perivascular immunoreactive nerve fibers were localized around the aorta, coronary arteries and their branches down to the terminal vasculature. Arterioles in the atria showed greatest density of immunoreactive varicosities of all blood vessels. The epicardium, endocardium and the conductive system also contained numerous CGRP-IR nerve fibers. In the myocardium the number of immunoreactive varicosities was variable. Many were present in both atria, moderate amounts were seen in the right ventricle and parts of the intraventricular septum, and only a few occurred in the left ventricle. CGRP-IR was infrequently found within intracardial ganglionic cells but was abundantly distributed in the surrounding nerve fibers.

Noradrenergic innervation of the cerebellar cortex in normal and in Purkinje cell degeneration mutant mice: Evidence for long term survival following loss of the two major cerebellar cortical neuronal populations

Purkinje cell degeneration mutant mice were examined during the course of Purkinje cell death (26 and 35 days old) and at 3, 5, 9 and 12 months of age. Glyoxylic acid fluorescence histochemistry for catecholamines was used to investigate possible alterations or reorganization of the noradrenergic fibers from the coeruleo-cerebellar system in response to the degeneration of two major cell types in the cerebellar cortex, of which one, the Purkinje cell, is reported to be the major target neuron. In control mice, noradrenergic fibers traveled in linear and tortuous profiles through the granule cell layer, formed pericellular arrays alongside Purkinje cell somata, and branched profusely into both radially oriented and longitudinally oriented chains. The density of noradrenergic varicosities diminished in the molecular layer with age. In the mutants, concomitant with the progressive shrinkage of the molecular layer, there was a progressive increase in the density of noradrenergic varicosities. This was most conspicuous at 9 and 12 months of age, at which time the molecular layer has been depleted not only of Purkinje cell dendrites, but also of parallel fibers. Noradrenergic fibers in these zones formed dense parallel bundles of varicose profiles whose density reached 621.3 ± 122.8% (mean ± SD, n = 4) at 9–12 months of age, compared with age-matched controls. Neurochemical measurement of norepinephrine content in whole cerebellum of the Purkinje cell degeneration mutants revealed no change compared with age-matched controls. We conclude that noradrenergic innervation persists in the cerebellar cortex despite the death of Purkinje cells and most of the granule cells. Although we found an increased density of varicosities in the molecular layer of mutant mice, progressing with age, we believe that this can be explained on the basis of the resultant geometry of the altered cerebellar cortex. It appears that the health of the environment surrounding the noradrenergic fibers in cerebellar cortex has little influence on their anatomical integrity.

Morphology of retinogeniculate terminals in the turtle, Pseudemys scripta elegans.

The dorsal lateral geniculate complex in turtles receives a bilateral, topographic projection from the retina and projects to the telencephalon. This study examined the morphology of individual retinogeniculate terminals that were filled with horseradish peroxidase by injections in the optic tract or optic tectum. A large number of retinogeniculate terminals were successfully filled and detailed drawings were prepared of 87 terminals. Terminals were classified into three types based on the size and number of varicosities in the terminal, and (if a terminal formed a spatially restricted arbor) the volume of the arbor. Type I retinogeniculate terminals form spatially restricted, large-volume arbors with a low density of large varicosities. Type II retinogeniculate terminals form small volume arbors with a high density of small varicosities. Type III retinogeniculate terminals, in contrast to types I and II, do not form spatially restricted arbors. Rather, they consist of sparsely branched axons that parallel the optic tract and contain scattered en passant varicosities. Plots of the distribution of different terminal types throughout the geniculate complex show that all three terminal types occur throughout the rostrocaudal and mediolateral extents of the complex. However, each terminal type has a preferential distribution with type II terminals being concentrated in the outer half of the neuropile, type I terminals in the inner half of the neuropile, and type III terminals in the cell plate. All three types can arise from axons that continue caudally to terminate in the tectum. These findings raise the possibility that various classes of retinal ganglion cells differ in their mode of termination within the geniculate complex, but the precise relation between the three types of retinogeniculate terminals and the classes of ganglion cells remains to be determined.

Ultrastructural immunocytochemical study of the dopaminergic innervation of the rat lateral septum with anti-dopamine antibodies

The dopaminergic innervation of the rat lateral septum has been investigated at ultrastructural level by immunocytochemistry using the unlabelled peroxidase-anti-peroxidase method with antidopamine antibodies. The specificity of the reaction has been carefully checked by immunological and histochemical controls. A strong immunoreaction was observed in fibres of the lateral septum as well as in their cells of origin in the ventral tegmental area. In the lateral septum, dopamine-immunoreactive fibres were localized in two distinct areas. A first area, located ventrally in the anterior part of the septum was characterized by a high density of immunoreactive varicosities with barely visible intervaricose segments. A more dorsal area, extending throughout the anteroposterior region of the septum, was characterized by immunoreactive fibres in pericellular arrangements. Electron microscopic observations revealed no difference in the ultrastructure of dopamineimmunoreactive profiles in the different areas. Reaction product was found in vesicles, linked to microtubules and in the cytoplasm. Three types of vesicles were seen: (i) small vesicles (30–50 nm) with varying intensity of immunoreaction, filling up the varicosities; (ii) rare large clear vesicles (50–80 nm) with no internal immunoreaction; (iii) very rare large dense vesicles (50–100 nm) with a strong dopamine immunoreactivity. Labelled profiles were observed in clearly defined asymmetrical synaptic contacts with somata and dendrites. Due to the lack of previous work dealing with the use of anti-dopamine antibodies for electron microscope immunocytochemistry, our observations are compared to previous data obtained by more indirect labelling techniques.

Ultrastructural studies on catecholaminergic terminals and GABAergic neurons in nucleus tractus solitarius of the rat medulla oblongata

Synaptogenesis of catecholamine (CA) boutons in the nucleus tractus solitarius (NTS) was compared between spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats at different ages. On the average, there were about 32 CA varicosities per 2200 μm 2 area of the NTS in both SHR and WKY rats as revealed by glyoxylic acid fluorescence microscopic (FM) morphometric study. The FM analysis indicated that there were no significant changes in the CA varicosity density between SHR and WKY rats. The CA boutons were labeled with 5-hydroxydopamine and appeared to contain small granular vesicles at the electron microscopic (EM) level. A total of 1402 CA boutons were studied in a 540, 000 μm 2 area of the NTS. The number of CA boutons involved in synaptic contacts vs the number of total CA boutons was used to obtain synaptic frequency which was taken as an index for synaptogenesis. A reduction of approximately 18% and 14% of synaptogenesis of CA boutons was observed in the NTS of SHR rats at 4 weeks (prehypertensive stage) and 12 weeks (early hypertensive stage) of age respectively, as compared to age-matched WKY rats. No significant difference of synaptogenesis of CA neurons was found between SHR and WKY rats at 16 weeks of age, a stage in which hypertension is well established and maintained in SHR rats. These results suggest that CA neurons with fewer synaptic contacts in the NTS may play a more important role in the initiation than in the maintenance of hypertension in the SHR rats. In addition to CA terminals, there were numerous GABAergic cell bodies in the NTS which were identified by immunocytochemistry using antibodies to the GABA synthesizing enzyme, l-glutamate decar☐ylase (GAD). GABAergic dendrites with GAD-positive reaction were often seen to receive several GAD-negative synapses at EM random profiles. In the text, a viewpoint is thus discussed that emphasizes that a synaptic abnormality of CA terminals with fewer synaptic contacts affecting GABAergic neurons may participate in the pathogenesis of hypertension. However, it remains to be determined as to whether or not there is a direct contact between CA boutons and GABAergic dendrites.

The distribution and morphological characteristics of the intracortical VIP-positive cell: An immunohistochemical analysis

Using a sensitive immunohistochemical procedure, we have undertaken a detailed morphological characterization of the vasoactive intestinal peptide-positive (VIP-positive) neuron in the cerebral cortex of the rat. VIP-positive neurons are present in all regions of cortex, and are usually strongly bipolar, possessing long, radially directed processes with very limited branching in the tangential plane. The most extensive dendritic branching occurs in layers I and deep IV-superficial V, and the density of axonal varicosities is highest in layers II–IV. In the visual cortex, approximately 50% of the labeled cell bodies are in layer II and III and 80% of the labeled cell bodies are contained in layers I–IV (superficial 600 μm of cortex). In order to determine the density and 3-dimensional distribution pattern of these cells, we prepared serial tangential sections through the rat visual cortex, mapped the distribution of all labeled cells in each section on transparent acetate sheets, and compressed these superimposed maps. This analysis demonstrated that: (1) approximately 1% of the cortical neurons are VIP-positive, (2) their distribution is fairly uniform and statistically random, (3) there are no large areas (i.e. with a diameter greater than 100 μm) that lack a VIP-positive cell, (4) on the average, there is one VIP-positive cell per column of 30 μm diameter, and (5) the average nearest neighbor distance on the compressed display is 15 μm. Given the morphological characteristics of VIP-positive cells, these data indicate that each VIP-containing cell is identified with a unique radial volume, which is generally between 15 and 60 μm in diameter, and overlaps with the contiguous domains of neighboring VIP-positive cells. These morphological data support the notion that VIP-containing neurons play an important functional role within radially oriented columns of cerebral cortex.

Serotonin nerve fibers in the primary visual cortex of the monkey. Quantitative and immunoelectronmicroscopical analysis.

A quantitative and immunoelectronmicroscopical analysis of serotonin nerve fibers in the primary visual cortex of the monkey (Macaca fuscata) was made using a sensitive immunoperoxidase method for serotonin. The overall numerical density of serotonin-containing varicosities in the primate striate cortex was approximately 770,000/mm3 and the highest concentration of immunoreactive varicosities (ca. 1,400,000/mm3) was observed in the upper portion of layer IVc, the next highest concentration being in layer IVb (ca. 1,180,000/mm3). At the ultrastructural level, the electron dense immunoreactive products were observed in the small granules (10-65 nm in diameter). The varicosities were usually small (0.5-1.0 micron in diameter) and made contact with both stellate and pyramidal cells. Serotonin fibers were often in close apposition to the poorly myelinated axons in layers IVb, V, and VI, and they rarely formed distinct synaptic structures with unlabelled neuronal elements.